1. Field of the Invention
The present invention relates to methods of preparation of avirulent live bacterial vaccines. Particularly, the present invention relates to attenuated live bacterial vaccines of the Pasteurellaceae family. More particularly, the invention is directed to methods of manufacturing live attenuated bacterial vaccines by the alteration of DNA adenine methylase (Dam) expression in the veterinary pathogens Pasteurella multocida, Mannheimia haemolytica, Actinobacillus pleuropneumoniae, Haemophilus somnus, Actinobacillus suis, and Haemophilus parasuis. More particularly, the inventors have discovered methods of manufacturing live attenuated bacterial vaccines wherein the alteration of DNA adenine methylase (Dam) expression in selected pathogens can be accomplished, for example, by 1) placing the Pasteurellaceae dam gene on a plasmid under the control of a promoter that causes increased dam expression in the particular bacteria species and results in live attenuated bacterial vaccines, 2) altering the chromosomal promoter for the dam gene so as to alter the expression of Dam in any of the above listed pathogenic bacteria, or 3) mutation of the dam gene so as to alter the expression of Dam in any of the above listed pathogenic bacteria. By altering Dam expression the bacteria is attenuated and suitable for use in live attenuated bacterial vaccines.
2. Background of the Technology
Livestock production is a major part of the world's agriculture economy. Disease annually depletes livestock populations and increases production costs. Either by prevention or post-infection treatment, the goal of limiting the impact of disease on livestock production is of great economic importance. Prior to the introduction of antibiotics, the practice of culling diseased animals and destroying them to prevent the spread of disease was, in many cases the only option open to the livestock producer.
During the past half century, the use of antibiotics has become vital for the treatment of bacterial infections in the livestock industry. The emergence of antibiotic resistance as a serious problem in human medicine has prompted concern about the public health implications of antibiotic use in agriculture. Antibiotics have been used in farm animals for three main reasons: 1) therapy to treat an identified illness, 2) prophylaxis to prevent disease in animals, and 3) performance enhancement to increase feed conversion, animal growth rate, or yield per animal.
Prevention of disease is always preferable to treatment of disease because treatments can be ineffective, costly, and due to the possibility of antibiotic resistance potentially unhealthful for consumers. The use of antibiotics as a preventive measure can also include the undesirable overuse of antibiotics.
Immunization of livestock against the effect of infectious diseases can be more effective than attempts to protect livestock with nonspecific antibiotic treatments. Conventional methods of immunization require the preparation of a vaccine using killed or weakened infectious organisms or agents. All too frequently, immunization failures in livestock might be attributed to one or more failures in the immunization process. The chemical or physical manipulations required for production of killed bacterial vaccines may alter the effectiveness of the immune response, causing vaccine failure. The immune response is very specific and the vaccine may contain organisms of the same family as the target disease, but if they are not of the same serotype (type within the family), the results may be disappointing. Vaccines can also lack the necessary potency or purity needed to properly stimulate an immune response in the vaccinated animal. Other problems commonly encountered in the vaccination of livestock may be outdated vaccine, improper handling or mixing of vaccines, or other delivery failures. For a variety of reasons, conventional vaccination of livestock can sometimes have disappointing results. For this reason, many livestock producers continue to rely on antibiotics as an infectious disease preventive measure.
There exists therefore a need to provide an effective method of preventing disease in livestock without the potential health hazard that can be associated with the prophylactic use of antibiotics in otherwise healthy livestock. Effective vaccines would be more economical and would avoid the potential for unnecessary antibiotic usage.
The concept of the present invention includes the preparation of attenuated live bacterial vaccines for a wide variety of pathogenic bacteria. Of particular interest to the inventors are the livestock diseases caused by veterinary pathogens Pasteurella multocida, Mannheimia haemolytica, Actinobacillus pleuropneumoniae, Haemophilus somnus, Actinobacillus suis, and Haemophilus parasuis. 
The association of Pasteurella multocida with bovine respiratory disease (BRD) has been well known since the early 1950's1. In most survey studies of feedlot BRD, Mannheimia (Pasteurella) haemolytica has been the most commonly isolated species, followed closely by Pasteurella multocida, with fewer cases of Haemophilus somnus2-4. However, in some studies, P. multocida is the most common isolate5,6, and many BRD investigators have become convinced that P. multocida is an important primary pathogen in BRD4,7. In young dairy calves, P. multocida is the most frequently isolated species from cases of pneumonia8,9. P. multocida is a commensal occupant of the upper respiratory tract of cattle5; the induction of disease is often associated with stress, especially from transportation. Potential virulence factors include polysaccharide capsule10, lipopolysaccharide,11,12, iron-regulated outer membrane proteins13,14, proteases15,16, neuraminidase16,17, and porins17,18. Recently, signature tagged mutagenesis was employed to identify 25 genes that, when inactivated, reduce virulence in the mouse intraperitoneal model19. The predicted gene products fell into 4 categories: regulatory, biosynthetic, known virulence factors, and unknown or novel. The whole genome sequence of an avian P. multocida isolate was published in 200120, which allowed identification of 104 potential virulence-related genes. However, the mechanisms that control expression of these potential virulence factors have not been determined.
DNA adenine methylase (Dam) is an important virulence gene regulator in the Enterobacteriaceae. In E. coli, Dam regulates transcription of several pili operons, including the pap (pyelonephritis-associated pili)21,22, sfa (S pili), fae (K88 pili), and daa (F1845 pili) operons23, and it regulates expression of a major outer membrane protein (Ag43)24. A Salmonella enterica serovar Typhimurium dam mutant strain had altered expression of more than 20 in vivo-induced (ivi) genes (elevated by 2- to 18-fold in a dam inactivated mutant compared to a wild type dam positive strain)25.
In bacterial species that possess a dam gene, alteration of dam expression causes substantial attenuation, as well as enhanced, protective antigenicity 25. A S. enterica serovar Typhimurium dam mutant had a LD50 that was >104 higher than wild type parent strain, and it was effective as a live attenuated vaccine after a single oral dose25. An overexpressing dam strain was also highly attenuated in mice 25. In Yersinia pseudotuberculosis and Vibrio cholerae, inactivation of the dam gene was shown to be a lethal mutation26. However, plasmid-mediated overexpression of the dam gene in Y. pseudotuberculosis resulted in a >6000-fold increase in LD50 in mice compared to wild type and a 5-fold defect in colonization of V. cholerae in a suckling mouse model compared to wild type26.
Although dam genes have been identified in the Pasteurellaceae as a result of genome sequencing projects20,27, there have been no reports of functional characterization of dam in any of these species.